1. Field of the Invention
This invention relates to a process for the highly specific modification of the genome of the yeast Candida tropicalis. This invention also relates to C. tropicalis strains with multiple POX4 and POX5 gene disruptions and to a method of using these strains for the production of dicarboxylic acids.
2. Description of the Related Art
Aliphatic dioic acids are versatile chemical intermediates useful as raw materials for the preparation of perfumes, polymers, adhesives and macrolid antibiotics. While several chemical routes to the synthesis of long-chain alpha, omega dicarboxylic acids are available, the synthesis is not easy and most methods result in mixtures containing shorter chain lengths. As a result, extensive purification steps are necessary. While it is known that long-chain dioic acids can also be produced by microbial transformation of alkanes, fatty acids or esters, chemical synthesis has remained the preferred route, due to limitations with the current biological approaches.
Several strains of yeast are known to excrete alpha, omega-dicarboxylic acids as a byproduct when cultured on alkanes or fatty acids as the carbon source. In particular, yeast belonging to the Genus Candida, such as C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. maltosa, C. parapsilosis and C. zeylenoides are known to produce such dicarboxylic acids (Agr. Biol. Chem. 35: 2033-2042 (1971)). Also, various strains of C. tropicalis are known to produce dicarboxylic acids ranging in chain lengths from C.sub.11 through C.sub.18 (Okino et al., In BM Lawrence, BD Mookherjee and BJ Willis (eds), Flavors and Fragrances: A World Perspective. Proceedings of the 10.sup.th International Conference of Essential Oils, Flavors and Fragrances, Elsevier Science Publishers BV Amsterdam (1988); and are the basis of several patents as reviewed by Buhler and Schindler, in Aliphatic Hydrocarbons in Biotechnology, H. J. Rehm and G. Reed (eds), Vol. 169, Verlag Chemie, Weinheim (1984).
It has been established that hydrocarbon substrates are enzymatically oxidized in the yeast microsomes. Following transport into the cell, n-alkane substrates for example, are hydroxylated to fatty alcohols by a specific cytochrome P450 system (Appl. Microbiol. Biotechnol., 28, 589-597 (1988)). Two further oxidation steps, catalyzed by alcohol oxidase (Kemp et al., Appl. Microbiol. and Biotechnol, 28, p370-374 (1988)) and aldehyde dehydrogenase, lead to the corresponding fatty acid. The fatty acids can be further oxidized through the same pathway to the corresponding dicarboxylic acid. The omega-oxidation of fatty acids proceeds via the omega-hydroxy-fatty acid and its aldehyde derivative, to the corresponding dicarboxylic acid without the requirement for CoA activation. However, both fatty acids and dicarboxylic acids can be degraded, after activation to the corresponding acyl-CoA ester, through the .beta.-oxidation pathway in the peroxisomes, leading to chain shortening. In mammalian systems, both fatty acid and dicarboxylic acid products of omega-oxidation are activated to their CoA-esters at equal rates and are substrates for both mitochondrial and peroxisomal .beta.-oxidation (J. Biochem., 102, 225-234 (1987)). In yeast, .beta.-oxidation takes place solely in the peroxisomes (Agr. Biol. Chem., 49, 1821-1828 (1985)).
The dicarboxylic acids produced through fermentation by most yeasts, including C. tropicalis, are most often shorter than the original substrate by one or more pairs of carbon atoms and mixtures are common (Ogino et al., 1965; Shio and Uchio, 1971; Rehm and Reiff, 1980; Hill et al., 1986). This is due to the degradation of the substrate and product by the peroxisomal .beta.-oxidation pathway. This series of enzymatic reactions leads to the progressive shortening of the activated acyl-CoA through the cleavage of 2 carbon acetyl-CoA moieties in a cyclic manner. The initial step in the pathway, involving oxidation of the acyl-CoA to its enoyl-CoA derivative, is catalyzed by acyl-CoA oxidase. The enoyl-CoA is further metabolized to the .beta.-keto acid by the action of enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase as a prerequisite to the cleavage between the alpha- and beta-carbons by 3-ketoacyl-CoA thiolase. Mutations causing partial blockage of these latter reactions result in the formation of unsaturated or 3-hydroxy-monocarboxylic or 3-hydroxy-dicarboxylic acids (Meussdoeffer, 1988). These undesirable by-products are often associated with biological production of dicarboxylic acids. It is also known that the formation of dioic acids can be substantially increased by the use of suitable mutants (Shiio and Uchio, 1971; Furukawa et al., 1986; Hill et al., 1986; Okino et al., 1986). The wild-type yeasts produce little if any dicarboxylic acid. Often, mutants partially defective in their ability to grow on alkane, fatty acid or dicarboxylic acid substrates demonstrate enhanced dicarboxylic acid yields. However, these mutants have not been characterized beyond their reduced ability to utilize these compounds as a carbon source for growth. In all likelihood, their ability to produce dicarboxylic acids is enhanced by a partial blockage of the .beta.-oxidation pathway. Furthermore, compounds known to inhibit .beta.-oxidation (ie. acrylate) also result in increased dicarboxylic acid yields (Zhou and Juishen, 1988).
Therefore, it would be desirable to have an effective block of the .beta.-oxidation pathway at its first reaction, catalyzed by acyl-CoA oxidase. A complete block, here, should result in enhanced yields of dicarboxylic acid by redirecting the substrate toward the omega-oxidation pathway while preventing reutilization of the dicarboxylic acid products through the .beta.-oxidation pathway. In addition, the use of such a mutant should prevent the undesirable chain modifications associated with passage through .beta.-oxidation, such as unsaturation, hydroxylation, or chain shortening. No mutants obtained by random mutagenesis are yet available in which this enzyme has been completely inactivated. While the C. tropicalis acyl-CoA oxidase genes have been cloned and sequenced (Okazaki et al., 1986) the lack of a method for the targeted mutagenesis of the C. tropicalis genome has prevented specific inactivation of the chromosomal acyl-CoA oxidase genes. A method for targeted gene disruption in yeast of the genus Pichia has been disclosed in European Patent Application 0 226 752. However, the present invention is the first description of targeted mutagenesis in C. tropicalis.
The production of dicarboxylic acids by fermentation of unsaturated C.sub.14 -C.sub.16 monocarboxylic acids using a strain of the species C. tropicalis is disclosed in U.S. Pat. No. 4,474,882. The unsaturated dicarboxylic acids correspond to the starting materials in the number and position of the double bonds. Similar processes in which other special microorganisms are used are described in U.S. Pat. Nos. 3,975,234 and 4,339,536, in British Patent Specification 1,405,026 and in German Patent Publications 21 64 626, 28 53 847, 29 37 292, 29 51 177, and 21 40 133.
None of the processes mentioned above gives the desired dicarboxylic acids in quantities sufficient to be commercially viable.